Endopeptidases are proteolytic peptidases that cleave peptide bonds between internal amino acids in proteins. These enzymes are classified as proteases. Proteases, including peptidases, are found ubiquitously in prokaryotes and eukaryotes. For example, at least 16 superfamilies of serine proteases have been described, each containing many families of proteins with evidence of convergent evolution in their protein structure. Serine proteases have been used in an industrial context, for example, in detergents. In this context, serine protease have been mutated to increase their thermostability (see for example, U.S. Pat. Nos. 4,980,288, 5,246,849 and 5,340,735). Serine proteases have also been used for molecular biology applications, for example, in the preparation of nucleic acid samples from cell isolates. To this end, a highly thermostable Proteinase K (also referred to as an endopeptidase K belonging to the peptidase family S8) from Engyodontium album (Tritirachium album) is commercially available and has a broad specificity for breaking down proteins with activity over a wide temperature range. In many applications, Proteinase K should be completely removed from a sample following its use and prior to subsequent reactions, for example, reactions using nucleic acid enzymes. Indeed, the use of Proteinase K in multi-step enzymatic workflows is adversely affected by its relative thermostability, where the temperatures required to fully inactivate Proteinase K often adversely affect the substrate (nucleic acid) structure. For example, temperatures above 65° C. result in partial denaturation of double stranded DNA that can result in DNA damage. Moreover, temperatures above 60° C. can result in hydrolysis of RNA (see e.g. Tenhunen, Mol. Cell Probes., 1989, 3(4): 391-396).
While this problem can be solved in-part by purifying DNA or RNA away from the Proteinase K after treatment using beads or columns, many workflows, e.g., those that involve low abundance DNA or RNA samples, cannot afford the sample loss that occurs when separation methods are used.